Pigs, and in particular members of the species Sus scrofa, are invaluable as a model organism. Because both the size and anatomy of domesticated pigs closely resembles that of humans, they are used in the research of a variety of important human diseases. However, the existing transgenic models of severe combined immunodeficiency disease (SCID) cannot mimic any of the known human immunodeficiency diseases where conditioning for bone marrow transplants includes irradiation. Further, none of the transgenic models represent a natural porcine model of SCID, and none of the transgenic models target the Artemis gene or any gene which causes radiosensitive immunodeficiency. Thus these transgenic models cannot mimic any of the known human immunodeficiency diseases where conditioning for bone marrow transplants includes irradiation.
SCID is a group of primary immunodeficiency disorders characterized by increased susceptibility to severe infections. SCID is caused by heritable defects of the cellular and humoral immune system, which result in a number of different phenotypes. Individuals with SCID have low numbers of circulating lymphocytes, T cell lymphopenia, and variable defects in B and NK cell populations. As a result, individuals with SCID have increased susceptibility to infections and increased mortality, commonly characterized by a severe deficiency in naïve T-cells. Infants born with SCID typically appear normal at birth, but are at high risk of serious infections after waning of maternal antibodies. If untreated, SCID has 100% mortality. Treatment is generally hematopoietic stem cell transplantation (HSCT), although gene therapy has been successfully used in some forms of SCID.
Characterization of the molecular mechanisms of SCID is integral to the development of diagnostic assays and treatments for the disease. A number of animal models for SCID exist, including several mouse models. However, murine and other small animal models often translate poorly to human disease due to important differences between mice and humans, including differences in immune response molecules and networks. Domestic pigs are much closer to humans, both physiologically and immunologically, and therefore a SCID pig represents a much better model for both the immune-compromised patient and for cancer and stem cell research, but such models have been lacking. While several reports have used molecular technologies to mutate one or more of several genes to cause a SCID phenotype in pigs, these reports are mainly limited to describing the phenotype of the mutated pigs. Further, these mutations are limited to genes that do not cause immunodeficiency syndromes that include a sensitivity to irradiation, as demonstrated for at least six types of human SCID genetic disease Previous work has also demonstrated that human cells transferred into SCID pigs are not destroyed due to lack of an immune system, but a practical model for use in exploiting SCID pigs for vaccine and other biomedical research has not been reported.
The Artemis gene—also referred to as the DNA cross-link repair 1C (DCLRE1C) gene—encodes a nuclear protein that is involved in V(D)J recombination and DNA repair. In pigs, based on build 10.2 of the swine genome, the Artemis gene is located on the forward strand of chromosome 10 and begins at approximately base number 51553277 and ends at approximately base number 51596761. Artemis includes 15 exons spanning 44 kb. The mRNA of porcine Artemis is 2388 nucleotides, encoding a protein that is 762 amino acids long. The protein has endonuclease activity on 5′ and 3′ overhangs and hairpins when complexed with and phosphorylated by DNA-dependent protein kinase catalytic subunit (PRKDC). Artemis is a member of the SNM1 family, which is defined by homology to yeast SNM1. Artemis is also referred to as SNM1C. Artemis is responsible for the resolution of hairpin coding ends in V(D)J recombination. In DNA double-strand break repair, Artemis is implicated in the end-processing step of the non-homologous end-joining (NHEJ) pathway. Artemis is the nuclease required for the resolution of hairpin coding ends during V(D)J recombination, the process by which B cell antibody genes and T cell receptor genes are assembled from individual V (variable), D (diversity), and J (joining) segments. For example, in joining a V segment to a D segment, the RAG (recombination activating gene) nuclease cuts both DNA strands adjacent to a V segment and adjacent to a D segment. The intervening DNA between the V and D segments is ligated to form a circular DNA molecule that is lost from the chromosome. At each of the two remaining ends, called the coding ends, the two strands of DNA are joined to form a hairpin structure. Artemis nuclease, in a complex with PRKDC, binds to these DNA ends and makes a single cut near the tip of the hairpin. The exposed 3′ termini are subject to deletion and addition of nucleotides by a variety of exonucleases and DNA polymerases, before the V and D segments are ligated to restore the integrity of the chromosome. The exact site of cleavage of the hairpin by Artemis is variable, and this variability, combined with random nucleotide deletion and addition, confers extreme diversity upon the resulting antibody and T-cell receptor genes, thus allowing the immune system to mount an immune response to virtually any foreign antigen.
In Artemis-deficient individuals, V(D)J recombination is blocked because the hairpin ends cannot be opened, and so no mature B or T cells are produced, resulting in SCID. Artemis was first identified as the gene defective in a subset of SCID patients that were unusually sensitive to radiation. Cells deficient in Artemis are more sensitive than normal cells to X-rays and to chemical agents that induce double-strand breaks (DSBs), and they show a higher incidence of chromosome breaks following irradiation. Artemis-deficient patients are also among those patients with poorer than average outcomes after bone marrow transplantation, thus a good large animal model that would recapitulate these defects would be useful for testing better methods of bone marrow transplant treatments.
The inventors have identified pigs with SCID, and a novel genetic basis of SCID in pigs. The inventors have further identified the genomic region that harbors the causative mutation, and have developed genetic marker tests that can be used to identify SCID pigs and SCID carriers. Specifically, the inventors provide a pig that possesses a mutated Artemis gene, resulting in the decreased production and/or function of the Artemis protein gene product.
It is an object of the present invention to provide the molecular basis for non-induced SCID in pigs.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects with SCID.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a model of human SCID for biomedical research.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a xenograft recipient in cancer research.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a xenograft recipient in stem cell research.
It is a further object of the present invention to provide porcine subjects and groups of porcine subjects to serve as a xenograft recipient in vaccine research.
It is a further object of the present invention to provide a genetic test for determining whether a porcine subject has SCID.
It is a further object of the present invention to provide a genetic test that can be used to identify affected pigs or carriers for the defect in pig populations, including commercial populations.
It is a further object of the present invention to provide a genetic test can be used to identify SCID affected piglets at an early age.
It is a further object of the present invention to provide a genetic test that can be used in biomedical research.
It is a further object of the present invention to provide further characterization of the SCID phenotype for animal disease and biomedical research, including research into the immune system, cancer research, the effects of disease, cell and tissue transplantation, and for testing of new vaccines and therapeutic agents for immuno-compromised individuals.
It is yet another object of the invention to provide further information for understanding SCID in pigs.
It is yet another object of the present invention to provide methods of identifying other mutations that are in linkage disequilibrium with or that are causative of SCID in specific lines, populations, or breeds of pigs.
It is a further object of the present invention to provide methods and compositions for curing, treating, alleviating, or inhibiting SCID.
It is a further object of the present invention to provide pharmaceutical compositions for curing, treating, alleviating, or inhibiting SCID.
Other objects will become apparent from the detailed description of the invention which follows.